Project Summary Cytokinesis is an essential step in cell division during which the newly separated genomes, cytoplasm, and organelles are partitioned from a mother cell into two daughter cells. How key events in cytokinesis are coordinated remains largely unknown because of the poor spatiotemporal resolution of each event and genetic redundancy. The long-term goal of our research is to elucidate the molecular mechanisms of cytokinesis. The objective of this application is to investigate the coordination of actomyosin contractile-ring constriction, plasma- membrane deposition, and extracellular septum formation during cytokinesis. The fission yeast Schizosaccharomyces pombe is a favorable model system for these studies because it is genetically tractable, has efficient homologous recombination facilitating gene targeting, and carries out cytokinesis using distinct temporal and spatial pathways. Previous studies on fission yeast cytokinesis and our solid preliminary data led to our central hypothesis that contractile-ring constriction guides plasma-membrane deposition and septum formation via vesicle trafficking pathways during cytokinesis. We will test this hypothesis by investigating three specific aims: we will characterize the molecular mechanisms that 1) cause contractile-ring constriction, 2) coordinate ring constriction and plasma-membrane deposition, and 3) coordinate ring constriction and septum formation. We will employ complementary approaches including genetics, quantitative microscopic imaging with high spatiotemporal resolution, biochemistry, and mathematical modeling in these studies. We will combine innovative approaches and hypotheses to investigate the three specific aims involving the most conserved aspects of cytokinesis. Our study on roles of four novel proteins Rng13, Rng14, Rng15, and Rng16 will provide molecular links among key cytokinesis events. These proposed studies are significant because they will advance our understanding of cytokinesis in three important ways: a) elucidating the basic principles and contributions of key actin-binding proteins in contractile-ring constriction; b) shifting the current paradigms on where and how vesicles are tethered during cytokinesis; and c) elucidating how glucan synthases at the division site coordinate ring constriction and septum formation for successful cytokinesis. Discerning molecular mechanisms that control proper completion and coordination of key events of cytokinesis in a simple model system is a critical step towards understanding more complicated but similar processes in human cells.